1. Field of the Invention
This invention involves the extraction, concentration and bioassay measurement of the concentration of parathyroid hormone in biological or other fluids. This application is related to U.S. Patent Application Ser. Nos. 442,649 filed Nov. 18, 1982 and 449,164 filed Dec. 13, 1982.
2. Description of the Prior Art
Human parathyroid hormone (hPTH) has a major role in maintaining the constancy (homeostasis) of calcium in cells and extracellular fluid, despite marked variations in calcium intake and excretion. It is now apparent that circulating PTH peptides fall into two groups: a common group and an unusual group. The common group consists of substantially intact hPTH believed to be hPTH.sup.1-84 and fragments thereof believed to be hPTH.sup.1-34 and hPTH.sup.35-84. Of these, hPTH.sup.1-84 and hPTH.sup.1-34 are believed to be biologically active, while hPTH.sup.35-84 is believed to be biologically inactive. Common group PTH peptides circulate in normal individuals and in those with primary and secondary hyperparathyroidism. The unusual group consists of a truncated form of substantially intact hPTH believed to be hPTH.sup.1-64 and fragments thereof believed to be hPTH.sup.1-34 and hPTH.sup.35-64. Of these, hPTH.sup.1-64 and hPTH.sup.1-34 are believed to be biologically active, while hPTH.sup.35-64 is believed to be biologically inactive. In parathyroid adenoma, tumors may secrete either or both groups, although clinical data indicates that secretion of the unusual group is quite common for this disorder.
There is increasing interest in measuring in human serum or plasma the concentration of the biologically active peptides of both the common group and the unusual group. Measurement of PTH in human serum or plasma by radioimmunoassay is well established, but, with the exception of the radioimmunoassay disclosed in U.S. Patent Application Ser. No. 449,164, such radioimmunoassays generally suffer from several disadvantages. One disadvantage is the difficulty of measuring the concentration of bioactive PTH in normal serum. The concentration of bioactive PTH in such cases is generally below the lower limit of the sensitivity of radioimmunoassays. Another disadvantage is that much of what is measured by radioimmunoassay is thought to be biologically inactive.
Sensitive and convenient bioassays for PTH, on the other hand, have not generally been available. Existing PTH bioassays utilize cultured animal tissue, cultured animal cells, or tissue extract containing adenylate cyclase-coupled PTH receptors. Certain PTH peptides bind the PTH receptors causing a complex response in the tissue, cells or extract which may be measured by the stimulated generation of cAMP. It can also be measured by the stimulation of glucose 6-phosphate dehydrogenase activity in the distal convoluted tubules of segments of kidney tissue maintained in vitro. Other means for measuring the stimulation of such PTH receptors are certainly possible in light of the complex response to such stimulation in the tissues, cells or extracts.
Bioassays directed to the stimulated generation of cAMP include the following. In F. Bringhurst, et al., "Metabolism Of Parathyroid Hormone By Kupffer Cells: Analysis By Reverse-Phase HPLC, "BIOCHEM, 21: 4252-4258 (1982), R. Zonefrati, et al., "Parathyroid Hormone Bioassay Using Human Kidney Cortical Cells In Primary Culture, "ACTA ENDOCRINOLOGICA, 100: 398-405 (1982), and R. Nissenson, et al., "Endogenous Biologically Active Human Parathyroid Hormone: Measurement By A Guanyl Nucleotide-Amplified Renal Adenylate Cyclase Assay," J. CLIN. ENDOCRIN. & METAB., 52: 840-846 (1981), all incorporated in their entirety herein by reference thereto, the adenylate cyclase activity of, respectively, cultured cells of rat osteogenic sarcoma, cultured cells of normal human kidney cortical plasma membranes, and homogenized extracts of canine renal cortical plasma membranes are measured by cAMP generation. These bioassays illustrate prior art attempts to overcome a basic problem in measuring adenylate cyclase activity by cAMP. The concentration of bioactive hPTH in serum is generally below the sentivity of such bioassays. This is especially so for serum taken from normal subjects, but is also pronounced for serum taken from renal dialysis patients where the concentration of bioactive PTH is only slightly elevated from undetectably low normal values.
The bioassay disclosed in Zonefrati, supra, illustrates the search for a more sensitive assay in the choice of tissue containing the adenylate cyclase activity. The bioassay disclosed in Bringhurst, supra, illustrates the search for a more sensitive assay in the choice of tissue and in using radioimmunoassay of cAMP. The bioassay disclosed in Nissenson, supra, illustrates the search for a more sensitive assay in amplifying adenylate cyclase activity and cAMP generation by incorporating Gpp(NH)p. These prior art attempts at increasing the sensitivity of bioassays measuring adenylate cyclase activity by cAMP generation are not wholly satisfactory, however, as evidenced by the lower limits of sensitivity claimed for these bioassays.
Bioassays measuring stimulation of glucose 6-phosphate dehydrogenase activity in the distal convoluted tubules of segments of kidneys are commonly referred to as "cytochemical bioassays." Cytochemical bioassays are believed to utilize the same receptor as the above discussed assays but measure stimulation of glucose 6-phosphate dehydrogenase activity rather than adenylate cyclase activity. Examples of such bioassays are disclosed in D. Chambers, et al., "A Sensitive Bioassay Of Parathyroid Hormone In Plasma," CLIN. ENDOCRINOLOGY, 9: 375-379 (1978) and S. Fenton, et al., "Preliminary Studies With The Sensitive Cytochemical Assay For Parathyroid Hormone," CLIN. ENDOCRINOLOGY, 9: 381-384 (1978). These cytochemical bioassays have respective sensitivities of 5 fg/ml and 0.1 fg/ml of bioactive PTH in serum samples. The sensitivity of these cytochemical bioassays reaches far below the concentration of bioactive PTH in normal serum. These cytochemical bioassays, however, suffer from a very serious disadvantage. As pointed out in Nissenson, supra, at p. 844, although the cytochemical bioassays are capable of detecting femptogram quantities of bioactive PTH in serum, these elegant bioassays are not practical for the routine assay of PTH in large numbers of samples. The cytochemical bioassay requires a remarkably high level of experimental and laboratory expertise of its practitioners. The cytochemical bioassay also requires a substantial amount of time and effort in performing the assay.
Measuring bioactive PTH peptides in human serum can prove to be an extremely helpful and effecient clinical tool in the differential diagnosis and management of hypercalcemia; most notably, it can help to diagnose tumors and hyperplasia of parathyroid glands. Measuring bioactive PTH peptides in human serum can also be useful for the diagnosis and management of hypocalcemia, which is usually reflected as subnoral blood concentratios of bioactive PTH peptides. PTH bioassay can also be used to assess the status of renal osteodystrophy in renal failure patients on chronic dialysis. The application of PTH bioassay in differential diagnosis and management of these disease states requires a bioassay with a sensitivity capable of measurements in the 10 picomole/liter range while also being capable of assaying large numbers of samples in a clinical setting. The prior art bioassays discussed above do not fulfill these requirements.